We have previously reported that cultured hepatocytes metabolize acetaminophen to glucuronide, sulfate and glutathione conjugates, even after 24-48 hours in culture. We have now characterized the relationship between acetaminophen activation and loss of plasma membrane function in these cultured cells. Correlations was observed between various parameters of plasma membrane injury e.g., leakage of cytoplasmic enzymes lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase and uptake of trypan blue. Moreover we have correlated these with the morphology of the cultured hepatocytes utilizing the transmission electron microscope. The injury was found to occur in certain cells and not others: thus exhibiting as all-or-none character. The omission of CaCl2 from the culture medium was found to alter the kinetics of LDH release but failed to prevent cell injury.